ABSTRACT Liver fibrosis, which results from chronic liver damage in conjunction with the accumulation of extracellular matrix (ECM) proteins, is characteristic of several chronic liver diseases. Dynamic changes to the liver microenvironment (LME) are widely recognized as a critical participant in liver fibrosis progression and therapeutic responses. LME components, including interactions between parenchymal (hepatocytes) and non- parenchymal cells (liver sinusoidal endothelial cells [LSECs], hepatic stellate cells, kupffer cells), signaling molecules (ligands-collagen, laminin), and mechanical cues from ECM, have been implicated in the progression of liver fibrosis. Stellate cells activation is the hallmark of liver fibrosis; however, the effect on hepatocytes and LSECs function has not been extensively understood. Also, the mechanisms by which the LME components regulate liver function and various signaling cascades are poorly understood, thus limiting the development of optimal diagnosis and treatment regimes for liver diseases (e.g., alcoholic liver disease, nonalcoholic fatty liver disease, non-alcoholic steatohepatitis, and hepatitis B and C). Therefore, there is a critical need to develop in vitro models that simulate the dynamic LME components and effectively study their role in liver fibrosis. To study the direct effects of LME on cell signaling, it is imperative to use in vitro liver models to simulate the fundamental complexity and dynamism of liver fibrosis and to achieve greater translational validity. The goal of this application is to use a multidisciplinary approach to develop three independent in vitro liver models to study how different LME components (hepatocytes-LSEC interactions, ECM stiffness, ligand type and density) regulate hepatocyte and LSEC function and what role these LME components play in the progression of liver fibrosis. The specific aims of the proposed study are to: 1) investigate the effect of hepatocytes-LSEC interaction(s) on hepatic function, 2) investigate how variation in stiffness alters hepatic cell function, and 3) determine the role of ligand type/density in regulating liver cell function. This work will provide a significant advancement in the ability to utilize in vitro liver models to accurately describe the liver function and metabolism in normal versus diseased states, and especially how the LME regulate the development and maintenance of liver function. Importantly, this model is innovative as it will chronologically emulate the fibrosis stage, is similar to clinical conditions, and boasts an environment that is more controlled and systematic than animal models. This project is expected to have a progressive impact on the study of liver fibrosis and related fields because the availability of a liver model that retains LME will facilitate understanding of the molecular mechanisms that underlie LME activities in mechanisms critical for the maintenance of liver biology.